1. Field of the Invention
This invention relates to hybridoma cell lines and monoclonal antibodies produced therefrom which may be used to detect nicarbazin.
2. Description of the Prior Art
Nicarbazin is a feed additive used to prevent outbreaks of cecal and intestinal coccidiosis in poultry, and is composed of an equimolar complex of 4,4xe2x80x2-dinitrocarbanilide (DNC) and 2-hydroxy-4,6-dimethylpyrimidine (HDP). Coccidiosis causes economic losses to the poultry industry [Long (Ed.), The Biology of the Coccidia, University Park Press, Baltimore, Md., 1982]. Nicarbazin was the first agent found to give satisfactory control of coccidiosis in broiler production (Cuckler et al., 1955, Science, 122:244-245; and Cuckler et al., 1956, Poult. Sci., 35:98-109). Nicarbazin also is used to increase the rate of weight gain. The complex, DNC-HDP, is ten times more potent in the control of Eimeria tenella, the primary coccidia cecal pathogen, as is DNC by itself. However, HDP when used alone was observed to have no anticoccidial activity (Cuckler et al., 1955, ibid). It was proposed that ultrafine crystals that are obtained as a result of complex formation allows for better dissolution of DNC resulting in improved anticoccidial activity (Rogers et al., 1983, Science, 222:630-632). Chickens also excrete DNC more slowly than HDP (Porter and Gilfilan, 1955, Poultry Sci., 34:995-1001). The U.S. Food and Drug Administration (FDA) has established a withdrawal period for nicarbazin of 4 days with a tolerance of 4 ppm (Code of Federal Regulations, 21 CFR 556.445). The Food Safety and Inspection Service (FSIS) uses high performance liquid chromatography (HPLC) with UV detection to analyze for DNC (Anon, in Analytical Chemistry Laboratory Guidebook: Residue Chemistry, USDA-FSIS Science and Technology Program, Washington, D.C., 1991, p. NIC-1). The FSIS method, as well as other published methods, use a variety of organic solvents (e.g., ethyl acetate, acetonitrile, hexane, dimethylformamide, and methanol) for extraction and other steps used in the analyses of DNC (Michielli and Downing, Jr., 1974, J. Agric. Food Chem., 22:449-452; Wood, Jr. and Downing, 1980, J. Agric. Food Chem., 28:452-454; Macy and Loh, 1984, J. Assoc. Off. Anal. Chem., 67:1115-1117; Parks, 1988, J. Assoc. Off. Anal. Chem., 71:778-780; and Lewis et al., 1989, J. Assoc. Off. Anal. Chem., 72:577-581); matrix solid-phase dispersion extraction followed by liquid chromatographic determination of DNC also has been used (Schenck et al., 1992, J. AOAC Intern., 75:659-662). All of these methods are time-consuming and result in generating substantial organic solvent wastes.
Our laboratory has had a long-term interest in developing monoclonal antibodies and immunochemical methods for the determination of all regularly used coccidiostats in poultry production. Detection methods based on immunoassays can greatly increase the rate of sample through-put, and allow the screening of increased numbers of samples without increasing the cost of analysis.
Our original approach to the production of monoclonal antibodies (MAbs) to DNC was to make a hapten from the drug DNC and use it for immunization. This was accomplished by using DNC and 4-hydrazinobenzoic acid to form the hydrazone. The hydrazone was then coupled to a carrier protein, keyhole limpet hemocyanin (KLH), using 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide HCl (EDC), and mice were immunized with the conjugate. Cell fusions were performed and screened for antibody activity. Antibodies to the hapten were obtained, but these were never observed to compete with xe2x80x9cfreexe2x80x9d DNC.
We have now discovered hybridoma cell lines which produce and secrete monoclonal antibodies which selectively bind to 4,4xe2x80x2-dinitrocarbanilide (DNC). We have unexpectedly found that these hybridomas may be obtained by using as an immunization agent or immunogen, p-nitroaniline which has been conjugated to an immunogenic carrier. DNC in biological samples may be detected and quantified by contacting the sample with the antibodies to form a DNC/antibody immunocomplex when DNC is present, which immunocomplex may then be detected. The monoclonal antibodies also may be incorporated into kits for the detection and quantification of DNC and/or nicarbazin.
It is an object of this invention to provide hybridoma cell lines that produce and secrete high affinity monoclonal antibodies which selectively bind to DNC.
Another object of this invention is to provide immunoassay methods for the measurement of DNC and/or nicarbazin in biological samples.
A further object is to provide kits useful for the assay of DNC and/or nicarbazin which include the monoclonal antibodies described herein.
Yet another object is to provide an immunization agent which may be used to produce hybridoma cell lines that produce and secrete high affinity monoclonal antibodies which selectively bind to DNC.
Other objects and advantages of this invention will become readily apparent from the ensuing description.